MELBOURNE, Australia – Researchers at the Murdoch Children’s Research Institute in Melbourne are pushing the boundaries on creating kidney tissue from stem cells.

For more than two decades, Melissa Little and her team at Murdoch have investigated the molecular and cell development basis of kidney disease and the potential for regeneration. The team has developed approaches for directing the differentiation of human pluripotent stem cells to human kidney organoids and is applying that knowledge to disease modeling, drug screening, cell therapy and tissue engineering.

Kidneys comprise about 1 million nephrons that filter the blood, and all nephrons are formed in utero. The number of those nephrons determines renal disease, Little said during the recent Ausbiotech conference.

Melissa Little, Theme Director of Cell Biology, Murdoch Children's Research Institute

A decade ago, the team sought to regenerate nephrons in kidneys via induced pluripotent stem cells (iPS) cells. She explained that nephron stem cells give rise to the kidney, and healthy humans have about 1 million nephrons. But those nephrons can’t be regrown; they need to be repaired.

The team studied mouse models to understand how cells differentiate into kidney cells, which she said are more related to patterning of tissues such as gonads in the blood. “We can track back development to gastrulation to the posterior primitive streak to the intermediate mesoderm, and that allows us to build a roadmap to walk through this process. We know the genes that should turn on and the signaling pathways required based on our understanding of development.”

The protocol is to take human iPS cells and grow them in a dish and walk them through the process of gastrulation.

“In two weeks, an organ forms through self-organization into a multicellular organ that patterns itself as the embryonic kidney would into a kidney organoid,” Little said.

Model of human kidney

This multicellular, highly patterned structure has glomeruli connected to proximal tubules and distal tubules and epithelium, and it contains roughly 100 nephrons.

The team conducted global RNA transcriptional profiling to compare the kidney organoids with whole human fetal tissues to confirm that the organ that most closely matched the organoid was a trimester one human kidney.

The team characterized cells in the model and identified 18 different cell types, including nephron progenitors and all the elements of the nephrons that arise from them.

“We can demonstrate they are robust, reproducible and transferable between lines, and the transcriptional profile is highly correlated with the human kidney,” Little said, noting that the organoids have evidence of collecting ducts, patterning nephrons, stroma and vasculature.

Going forward, the group is starting to collect iPS cells from patients that can be edited to correct mutations or add mutations.

“It’s the first time we have interrogated human development using CRISPR/Cas 9 gene editing to focus down on this process by developing a suite of reporter lines for characterizing cell types within kidney organoids,” she said.

“It’s not a process that can yet be moved into a pipeline,” Little said, “but it can be used to model inheritable kidney disease and, using disease modeling, to develop tissue types to improve diagnosis and understand mechanisms.”

Those tissue types can then be used to test different compounds on tissue models for drug screening and can also be engineered for new stem cell-derived tissue and organs for tissue regeneration.

Taking the research forward, a collaboration was formed with university hospitals across Australia, and the group, now called Kidgen, is working with nephrologists across Australia to recruit patients with kidney disease.

Kidgen is using those tools to identify cell types for disease modeling for diseases like childhood nephrotic syndrome, an inherited kidney disease that affects one in 15,000 children. “Roughly 50 percent will not get a genetic diagnosis, and for those with a diagnosis, we don’t understand the cause of the disease and have no specific treatment,” Little said. “The only treatment options are dialysis and/or transplants.”

It is also working with San Diego-based Organovo to develop a robotic algorithm to set up high-throughput screening.

Little said the size is an issue for the stem-cell derived model as the organoid has only 100 nephrons compared to the 1 million that are needed. The group is hoping to scale up microtissue production using 3D cellular printing.

More than 800 patients have been seen in Kidgen renal genetics clinics since the start of the collaboration. The network includes 19 nephrologists, 13 clinical geneticists and genetic counselors across Australia.

Roughly 200 participants have been recruited into the Melbourne genomics renal genetics flagship in Victoria. Called, HIDDEN (wHole genome Investigation to iDentify undetected Nephropathies), it is one of two renal genomics studies led by Kidgen and supported by Australian Genomics.

The clinical flagship will begin recruiting patients to its research study, providing whole genome sequencing to 200 adults and children with end-stage kidney disease of unknown cause.

The HIDDEN flagship will provide whole genome sequencing to eligible patients in an effort to reach a genetic diagnosis for their kidney disease. The study is one of only a few worldwide, sharing similar methods and partnerships with renal genomics studies in both England and New Zealand. Those studies aim to break new ground in kidney disease diagnosis and add to an emerging evidence base on the effectiveness of genomic techniques in nephrology.

Multidisciplinary teams will establish if genome testing will be appropriate for each patient and then proceed with patient enrollment. That process will be mirrored across sites in Australia, with DNA samples being sent to the Garvan Institute for testing.

While the HIDDEN study aims to assess the diagnostic utility of whole-genome sequencing for end-stage kidney disease of unknown cause, it is also undertaking secondary evaluations of cost-effectiveness and implications for pharmacogenomics.

Chronic kidney disease is increasing 6% per year, according to Kidney Health Australia. About 93,000 people are on transplant lists in the U.S., but only one in four people will receive a new organ. The rest are confined to dialysis, which leads to a poor quality of life, and 6% will have a mortality rate of five years.

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